Micro-oraganisms with glycosylation modulating action of intestinal cell surface

ABSTRACT

The invention concerns micro-organism strains, in particular of lactic acid bacteria, having a glycosylation modulating effect of intestinal cell surface. The invention also concerns a method for selecting micro-organism strains, in particular of lactic acid bacteria, which consists in measuring the average fluorescence intensity variation of HT29-MTX cells incubated in the presence of a lectin coupled with a fluorochrome after being in contact with the supernatant of the strain concerned. Said lactic acid bacteria strains can be used, optionally in the form of their active fraction, for preparing food compositions or medicines or food supplements, modulating glycosylation of glycoproteins of intestinal epithelial cells.

[0001] The invention concerns microorganisms having a modulatory effecton surface glycosylation or on the composition of sugars at the surfaceof intestinal cells, a method for selecting said microorganisms, andtheir uses in the food and medical sectors.

[0002] The gastrointestinal mucosa consists of a simple layer ofepithelial cells which are at least partially covered on the side of theintestinal lumen by a viscoelastic layer mainly consisting ofglycoconjugates. The epithelial cells synthesize the glycoconjugatespresent at their surface, which are the intermediates in numerousinteractions, in particular with lectins or adhesins, with bacterialtoxins, or with antibodies, bacteria, viruses, parasites. Theseglycoconjugates therefore constitute important intermediates in therelationship between the host and the intestinal flora.

[0003] These glycoconjugates are, as their name indicates, glycosylatedcompounds, that is to say on which are grafted sugar chains which arelong to a greater or lesser degree and which may be branched. In thehealthy human intestine, these sugars may be galactose (Gal), fucose(Fuc), N-acetylneuraminic acid or sialic acid, N-acetylglucosamine(GlcNAc), N-acetylgalactosamine (GalNac), linked to each other byvarious bonds. Three of them may be in a terminal position; they aregalactose, fucose and sialic acid.

[0004] A host and its intestinal microflora function as a complex systemin which the microflora has a significant impact on the host. In thecase of nonpathogenic or probiotic microorganisms, a symbiotic orcooperative relationship often exists between the host and themicroorganisms, the presence of the latter being necessary for goodbalance and good functioning of the host's intestine. By contrast, thepresence of pathogenic microorganisms, which is more rare, may havenegative consequences by preventing or reducing the presence ofprobiotic microorganisms, or even by having a parasitic action which isdirectly harmful to the host.

[0005] The fragile balance between the host and the microflora isdirectly linked to the intestinal environment and in particular to thequantitative and qualitative presence of the surface glycoconjugates.Indeed, it is known that some microorganisms, which may be probiotic orpathogenic, will be sensitive to certain sugars at the terminalposition. The intestinal bacteria can modulate the pattern ofglycosylation of the glycoconjugates present at the surface of theintestinal cells, without their mode of action being completelyelucidated as will be seen hereinafter. The bacteria can, on the onehand, induce the presence of such and such sugar during glycosylationand, on the other hand, break down the sugars present at the terminalposition of a chain, which qualitatively and/or quantitatively modifiesthe sugars present at the terminal position.

[0006] This modulation of the pattern of epithelial cell surfaceglycosylation causes modification of the intestinal environment, itbeing possible for the modified environment to promote the establishmentof certain microorganisms and/or to limit, or even avoid, theestablishment of other microorganisms. Modification of the environment,which may be generated by the microflora, therefore has a direct impacton the balance between the host and the microflora.

[0007] It would therefore be highly advantageous to have a model whichmakes it possible to rapidly and easily select microorganisms accordingto their action on the pattern of glycosylation. It would thus bepossible to identify microorganisms promoting the presence of one sugaror another, and therefore promoting the establishment of beneficialmicroorganisms such as, for example, probiotics and/or limiting theestablishment of pathogenic microorganisms.

[0008] Indeed, to the knowledge of the applicant, no microorganisms, inparticular lactic acid bacteria, have so far been identified which arecapable of finely acting accurately on the glycosylation pattern, andthus of accurately modulating this glycosylation pattern and thereforethe implantation of microorganisms.

[0009] Such microorganisms modulating the glycosylation pattern couldfind a use in particular in pharmaceutical or food compositions or foodsupplements. Indeed, they can make it possible to optimize thefunctioning of the intestinal cells and the good balance of themicroflora.

[0010] The document WO 99/29833 describes a novel bacterial strainhaving certain properties, for example an antimicrobial activity.However, the modalities of its action are not detailed, and inparticular the question of the possible action of the bacterium onsugars is never addressed.

[0011] Bry L. et al., in “A model of host-microbial interactions in anopen mammalian ecosystem”, Science, Vol. 273, pp. 1380-1383, Sep. 6,1996, studied in vivo (in mice) the influence of Bacteroidesthetaiotaomicron on fucosylation. Bacteroides thetaiotaomicron is partof the intestinal flora in humans and in mice. The model used isnevertheless limited to the study of fucosylation, with the exclusion ofother types of glycosylation. It is moreover an in vivo model, andtherefore longer and more complex to use than an in vitro model.

[0012] However, this model made it possible to emit the hypothesis ofthe existence of a soluble factor secreted by B. thetaiotaomicron, andwhich is thought to play the role of a signal bringing about themodification of surface glycosylation. This unidentified soluble factoris therefore thought to act without there being direct contact betweenthe bacteria and the target cells.

[0013] To the knowledge of the applicant company, no method has beendeveloped to date which makes it possible to select in vitro, easily andrapidly, various microorganisms, in particular various strains of lacticacid bacteria, according to their precise action on the pattern ofglycosylation of the intestinal epithelial cells, and more precisely onthe modulation or variation of the composition of each of the sugars.

[0014] In the context of the present application, the term “lactic acidbacteria” denotes bacteria capable of producing lactic acid, and inparticular the nonpathogenic bacteria chosen from the group comprisingStreptococcus, Lactobacillus, Lactococcus, Bifidobacterium andLeuconostoc, in particular B. breve, B. longum, L. lactis, S.thermophilus, L. casei, L. helveticus and L. bulgaricus.

[0015] A subject of the present invention is therefore such a method ofselection in vitro, which comprises the following steps:

[0016] bringing the culture supernatant for the strain ofmicroorganisms, in particular of lactic acid bacteria, into contact withcells of a cell line model representing the intestinal epithelial cells,

[0017] incubating,

[0018] extracting the cells and incubating with various lectins coupledto a fluorochrome,

[0019] washing,

[0020] measuring the mean fluorescence intensity (MFI) of each lectin,comparing with the measurements performed on a control sample in orderto evaluate the variation in MFI induced by the bacteria, selecting thestrain of microorganisms inducing a variation of at least 20% for atleast one sugar.

[0021] The latter step may in particular be carried out on plates, themeasurement of MFI being performed by a probe, according to techniquesknown to persons skilled in the art, or by flow cytometry. In the caseof flow cytometry, it is also possible to compare the graph obtained,representing the variation of fluorescence as a function of the numberof cells, with that obtained with a control sample, and to select thestrain of microorganisms inducing a variation in the shape of the graph.

[0022] Any cell line model representing intestinal epithelial cells maybe used in this method of selection. Persons skilled in the art willknow how to choose an appropriate cell line model among the knownmodels, for example the lines Caco-2 or HT29-MTX, which are cancerousintestinal epithelial cells in culture. These lines are considered asreproducing all or some of the mechanisms of regulation of the systemsin vivo and in particular the production of glycoconjugates. Any lineexhibiting this characteristic may be used in the method according tothe invention. As regards the HT29-MTX line, reference may be made toLesuffleur et al., “Growth adaptation to methotrexate of HT-29 humancolon carcinoma cells is associated with their ability to differentiateinto columnar absorptive and mucus-secreting cells”, 1990, Cancer Res.,50:6334-6343.

[0023] Centrifugation and filtration of the culture medium in which eachstrain of microorganisms to be tested has been incubated are carried outin order to extract the (“trial”) supernatant therefrom. Without wishingto be bound by any theory, the applicant considers that the supernatantcontains the soluble factor constituting the signal for modulatingglycosylation. The supernatant therefore constitutes an active fractionof a strain of microorganisms. Brought into contact with the intestinalepithelial cells, it will cause or not one or another modification ofglycosylation according to the strain of microorganisms tested.

[0024] Thus, in Bacteroides, the soluble factor was identified as beinga low molecular weight molecule (less than 8 kD) and that it isheat-sensitive. It can be assumed that the soluble factor from anotherstrain will exhibit similar, if not identical, characteristics.

[0025] The supernatant obtained from the culture medium which has notbeen in contact with the bacteria is used as a “control”.

[0026] An appropriate quantity of the “trial” and “control” supernatantsare incorporated into the culture medium in which the cells of the cellline model, for example HT29-MTX, are placed for an appropriate period.This period may be, for example, from 1 to 15 days.

[0027] In a particular embodiment, it is possible to add bacteria inplace of the supernatant. In this case, the bacteria may be added in theform of a suspension in the presence of penicillin G and of a strongbuffer making it possible to inhibit bacterial development withoutpreventing the viability of the bacteria, and acidification of themedium.

[0028] After this period of incubation, the cells of the cell line modelare detached and resuspended, and the same number of “trial” and“control” cells is incubated separately with a lectin of which thespecificity toward sugars (nature of the sugar, nature of the linkage)is known.

[0029] Each lectin, which is coupled to a fluorochrome, will bind to thesugar for which it is specific.

[0030] The “trial” and “control” cell suspensions are each washedseveral times, and the reactivity of each lectin is quantified byfluorescence measurements called MFI (mean fluorescence intensity).

[0031] This measurement may be carried out by any appropriate means, inparticular by means of flow cytometry (FACScan, Bencton-Dickinson) or bydirect fluorimetric measurements on plates.

[0032] When flow cytometry is used, a graph representing the variationof fluorescence as function of the number of cells is obtained for eachlectin. For each flow cytometry graph, the mean fluorescence intensitycorresponds to the median value.

[0033] Two comparisons are possible. On the one hand, regardless of themean of the measurement of fluorescence intensity, it is possible tocompare quantitatively the “trial” MFI with the “control” MFI. On theone hand, in the case of flow cytometry, it is also possible to comparequalitatively the “trial” result with the “control” result, and moreprecisely to compare the graph obtained with the “trial” suspension withthat obtained with the “control” suspension, that is to say to comparethe shape of the curves.

[0034] It is estimated that a decrease or an increase in this MFI of atleast about 20%, preferably at least about 28%, still more preferably atleast about 35%, reveals a significant change in the reactivity of eachlectin, and therefore of the sugar composition of the glycoproteinspresent at the surface of the epithelial cells.

[0035] This method therefore makes it possible to observe and toquantify in a simple and rapid manner the presence of each lectin on thesugar chains, and therefore to quantify the sugar whose lectin isspecific. It is then possible to compare the values obtained without andwith microorganisms, and thus to evaluate the glycosylation modulatingeffects of each strain of microorganisms, in order to select the strainshaving the desired effect: increase in such a sugar, decrease inanother.

[0036] The subject of the invention is also the strains ofmicroorganisms, in particular of lactic acid bacteria, capable ofmodulating the pattern of intestinal epithelial cell surfaceglycosylation, and more particularly the strains of microorganisms, inparticular of lactic acid bacteria, capable of being selected by themethod described above.

[0037] Such microorganisms therefore make it possible to modulateglycosylation, and therefore to reestablish or modify the intestinalenvironment in order to promote the establishment of probioticmicroorganisms and to avoid or limit the establishment of pathogenicmicroorganisms.

[0038] It is known, for example, that the receptor allowing adhesion ofEscherichia coli contains galactose and sialic acid; for Streptococcuspyogenes and Listeria monocytogenes, it is galactose; for Helicobacterpylori, it is fucose; and for Entamoeba histolytica, it is galactose andN-acetylgalactosamine.

[0039] The strains which are the subject of the invention may be inparticular the following strains: CNCM collection No. I-2492, CNCMcollection No. I-2493, CNCM collection No. I-2494 (CNCM=CollectionNationale de Cultures de Micro-organismes—Institut Pasteur—28, rue duDr. Roux—75724 Paris Cedex 15—France), deposited on Jun. 20, 2000.

[0040] It is possible to use such microorganisms, in particular suchlactic acid bacteria, or their active fraction, that is to say thefraction comprising the soluble factor, for the preparation of foodcompositions or medicaments or food supplements modulating intestinalepithelial cell surface glycosylation. These compositions may comprise asingle strain, or several strains, of microorganisms modulatingglycosylation.

[0041] Thus, compositions are available which make it possible tomaintain or reestablish a proper host-intestinal flora balance, or whichmake it possible to promote the establishment of one or another strainof microorganisms. They therefore make it possible to optimize thefunctioning of the intestinal cells and good balance of the microflora.It is possible in particular to use lactic acid bacteria, and toincorporate them into dairy products.

[0042] The strains tested in the trials below are given by way ofnonlimiting examples.

[0043] Lactic Acid Bacteria in Vitro Trials

[0044] Centrifugation and filtration of the culture medium in whichlactic acid bacteria (LAB) have been incubated overnight were carriedout in order to extract the (“trial”) supernatant therefrom.

[0045] The LAB strains used are the following: CNCM collection No.I-2492, CNCM collection No. I-2493, CNCM collection No. I-2494.

[0046] The supernatant obtained from the culture medium which has notbeen in contact with the bacteria is used as a “control”.

[0047] An optimum quantity for each “trial” and “control” supernatant(between 10% and 30%) is incorporated into the DMEM (“Dulbecco'sModified Eagle's Medium”) culture medium in which the HT29-MTX cell lineis placed for 5 to 15 days.

[0048] After this treatment, the HT29-MTX cells are detached andresuspended in a PBS solution (“Phosphate Buffered Saline Solution”),and the same number of “trial” and “control” cells is incubatedseparately with a lectin specific for a sugar (nature of the sugar,nature of the linkage) at optimum concentration for 30 minutes.

[0049] The commercial lectins used and their epitope(s) are thefollowing: Lectin Lectin Abbrevi- Name ation Epitope (s) Artocarpusintegrifolia Jak Gal Ricinus communis RCA I Gal Griffonia simplicifoliaGSI-B4 Galα3Gal I B4 Helix pomatia HPA GalNAc Datura stramonium DSAGlcNAc Wheat germ WGA GlcNAc and sialic acid Anguilla anguilla AAAα-L-fucose Ulex europaeus I UEA I Fucα2Galβ Maackia amurensis MAA sialicac. α 2,3 Sambucus nigra SNA sialic ac. α 2,6

[0050] All these lectins are marketed by EY Laboratories Inc. (SanMateo, Calif., USA) except RCA I, marketed by Sigma Aldrich(Saint-Quentin-Fallavier, France).

[0051] The following mean fluorescence intensity values are obtained byflow cytometry for each strain of lactic acid bacteria, and for the“control” and “trial” cells: CNCM I-2492 Lectin ″control″ ″trial″Variation RCA 424 392  −7.5% Jak 422 431  +2% DSA 237 241  +1.6% SNA 5748  −16% HPA 334 350  +4.5% CNCM I-2493 Jak 535 392 −26.7%  SNA 48 36 −25% HPA 354 379  +7% MAA 665 518  −22% WGA 1313 1376  +4.8% GSI 37 31 −16% RCA 317 247  −22% DSA 176 165  −6% CNCM I-2494 Jak 346 446  +29%SNA 34 35  +2.9% HPA 748 593 −20.7%  AAA 10 20 +100% MAA 668 644  −3.6%WGA 302 290  −4% GSI 30 32  +6.7% RCA 258 369  +43% DSA 176 198  +11%

[0052] Thus, the glycosylation modulating action of each bacterialstrain can be immediately observed. For example, it can be observed thatthe CNCM I-2494 strain causes an increase in Gal and α-L-Fucose, and adecrease in GalNAc.

1. A method of selecting strains of microorganisms comprising thefollowing steps: bringing the culture supernatant for the strain ofmicroorganisms into contact with cells of a cell line model representingthe intestinal epithelial cells, incubating, extracting the cells andincubating with various lectins coupled to a fluorochrome, washing,measuring the mean fluorescence intensity (MFI) of each lectin,comparing with the measurements performed on a control sample in orderto evaluate the variation in MFI induced, and selecting the strain ofmicroorganisms inducing a variation of at least 20% for at least onesugar, and/or comparing the graph obtained by flow cytometry,representing the variation of fluorescence as a function of the numberof cells with that obtained with a control sample and selecting thestrain of microorganisms inducing a variation in the shape of the graph.2. The method as claimed in claim 1, characterized in that the meanfluorescence intensity is measured by flow cytometry, and corresponds tothe median value of the graph obtained representing the variation offluorescence as a function of the number of cells.
 3. The method asclaimed in either of claims 1 and 2, characterized in that themicroorganisms are bacteria, in particular lactic acid bacteria.
 4. Themethod as claimed in any one of claims 1 to 3, characterized in that thestrain of microorganisms selected induces a variation in FI of at leastabout 28%, preferably at least about 35%.
 5. The method as claimed inany one of claims 1 to 4, characterized in that the cells of the cellline model are HT29-MTX cells.
 6. A strain of lactic acid bacteria,characterized in that it induces modulation of intestinal epithelialcell surface glycosylation.
 7. The strain of lactic acid bacteria asclaimed in claim 6, characterized in that it induces a variation of theshape of the graph obtained by flow cytometry and/or of the meanfluorescence intensity by at least about 20%, preferably by at leastabout 28%, still more preferably by at least about 25%, for at least onesugar, in a test comprising the following steps: bringing the culturesupernatant for the strain of bacteria into contact with cells of a cellline model representing the intestinal epithelial cells, in particularHT29-MTX cells, incubating, extracting the cells and incubating withvarious lectins coupled to a fluorochrome, washing, measuring the meanfluorescence intensity (MFI) of each lectin, and comparing with themeasurements performed on a control sample in order to evaluate thevariation in MFI induced, by the bacteria, and/or comparing the graphobtained by flow cytometry, representing the variation of fluorescenceas a function of the number of cells, with that obtained with a controlsample.
 8. A strain of lactic acid bacteria deposited under the numberCNCM I-2492.
 9. A strain of lactic acid bacteria deposited under thenumber CNCM I-2493.
 10. A strain of lactic acid bacteria deposited underthe number CNCM I-2494.
 11. The use of a strain of lactic acid bacteriacapable of being selected with the method as claimed in one of claims 1to 5, or of the active fraction of such a strain, for the preparation ofa food or pharmaceutical composition or a food supplement modulatingintestinal epithelial cell surface glycosylation.
 12. The use of astrain of bacteria as claimed in any one of claims 6 to 10, or of theactive fraction of such a strain, for the preparation of a food orpharmaceutical composition or of a food supplement modulating intestinalepithelial cell surface glycosylation.
 13. A food or pharmaceuticalcomposition, characterized in that it comprises at least one bacterialstrain as claimed in any one of claims 6 to 10 or at least an activefraction of one of these strains.
 14. The food or pharmaceuticalcomposition as claimed in claim 13, characterized in that it modulatesintestinal epithelial cell surface glycosylation.